Download The regulation of fatty acid biosynthesis in some

Journal of General Microbiology (1992), 138, 109-1 14. Printed in Great Britain
109
The regulation of fatty acid biosynthesis in some estuarine strains
of Flexibacter
PABLOINTRIAGO"
School of Ocean Sciences, Marine Science Laboratories, Menai Bridge, Gwynedd LL59 5E Y, UK
(Received 25 January 1991 ;revised 13 September 1991 ;accepted 3 October 1991)
The mechanism of unsaturated fatty acid biosynthesis in two estuarine strains of Ffexibucterwas investigated.
Addition of cyclic AMP (CAMP)inhibited the incorporation of radiolabelled acetate into fatty acids of lateexponential phase cultures of strain Inp2. Cerulenin selectively inhibited the incorporation of radioactive acetate
into the major fatty acid, 16:l. When Ffexibacterstrain Inp3 was grown in a medium of high osmotic strength,
polyunsaturated fatty acids were present, but they were absent from cultures grown in media with an osmotic
strength close to that of sea water. Addition of cAMP to culturesgrowing at high osmotic strength suppressed the
formation of polyunsaturated fatty acids; the uncoupler 2,4-dinitrophenol had a similar effect. The findings are
discussed in the context of the suggestion made by Intriago and Floodgate (Journal of General Microbiology 137,
1503-1509,1991) that Ffexibucterstrains possess both the anaerobic and aerobic pathways for unsaturated fatty
acid biosynthesis, the activity of the latter being modulated by the intracellular concentration of CAMP.
Introduction
Only two biochemical mechanisms are recognized for the
introduction of a double bond into a fatty acid. In the
anaerobic pathway, which can be considered part of the
fatty acid synthetase system, a double bond is formed
when the chain generally has 10 or 12 carbon atoms. The
chain is then lengthened to produce eventually either
C16 :1w7 or C16 :1w9 respectively (Goldfine & Bloch,
1961 ; Erwin & Bloch, 1964; Fulco, 1983). This pathway
is found in all anaerobes, facultative anaerobes and some
aerobic bacteria (Harwood & Russell, 1984; Schweizer,
1989). In contrast, the aerobic or oxygen-dependent
pathway introduces double bonds by desaturation of
existing chains, usually 16 or 18 carbon atoms in length.
This latter pathway is found in some aerobic bacteria,
especially Gram-positive ones, as well as non-parasitic
eukaryotes (Fulco, 1970; Schweizer, 1989). The position
of the first double bond in plants and microbes is usually
at the 9,lO position (Bloomfield & Bloch, 1960; Russell,
1978). Scheuerbrandt & Bloch (1962) suggested that the
Present address: Aqualab, PO Box 09-01-5738, Guayaquil,
Ecuador. Fax 4 284409.
Abbreviations: DNP, 2,4-dinitrophenol; UFA, unsaturated fatty
acid; PUFA, polyunsaturated fatty acid; p.p.t., parts per thousand
(salinity).
0001-6740 0 1992 SGM
anaerobic and the aerobic pathways were mutually
exclusive. However, Wada et al. (1989) have recently
demonstrated the presence of both pathways in a
psychrotrophic Pseudomonas.
The presence of polyunsaturated fatty acids (PUFAs)
in marine bacteria has been demonstrated (Delong &
Yayanos, 1986; Wirsen et al., 1987; Yazawa et al., 1988;
Intriago & Floodgate, 1991), but the mechanism controlling their biosynthesis is unknown. A potential regulator
such as cyclic AMP (CAMP) is known to affect cell
metabolism in different ways, including regulation of
gene expression (Pauli et al., 1974), control of cellular
morphogenesis (Larsen & Sypherd, 1974) and phosphorylation of proteins (Saha et al., 1988). In contrast,
there are few reports in the literature about the effect of
cAMP on fatty acid composition. Dallas et al. (1976)
showed that the fatty acid composition of Escherichia coli
was regulated by cAMP and Alaniz et al. (1976) found
that incubation of cultured mouse cells with cAMP
decreases the desaturation of linolenic acid.
Cerulenin was used in the present study because it is
known to reduce fatty acid synthesis in E. coli by
inhibiting /?-ketoacyl-ACP synthase (D'Agnolo et al.,
1973). Butkke & Ingram (1978) suggested that /?ketoacyl-ACP synthase I, which elongates cis-decenoylACP to C 16 :1w7 in E. coli, is more sensitive to cerulenin
than is /?-ketoacyl-ACP synthase 11, which elongates
C16 :1 to C18 :1. Thus, inhibition of unsaturated fatty
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110
P . Intriago
acid (UFA) synthesis by cerulenin can be considered as
an inhibition of the anaerobic pathway.
The rationale of this study was to examine the effect of
CAMP, cerulenin and high osmotic strength on the
incorporation of radioactive acetate into exponentially
growing cultures of Flexibacter strain Inp2, a PUFAcontaining strain, and to compare their effects on the
fatty acid composition of Flexibacter strain Inp3, which
is deficient in PUFAs.
Berthold HPLC radioactivity monitor (LB506C-1), connected to a
Compaq-Desk PRO 386/20 computer. A typical run time was 30 min.
Identification of peaks was based on comparison with pure standards,
and compared against gas-liquid chromatographic analyses.
The mobile phase used, acetonitrile/water (85 :15, v/v), did not
completely resolve C16:O from C18:1, and they were reported
together, but this did not affect the aims of this study.
It is relevant to point out that whereas fatty acid analysis by gasliquid chromatography agrees more closely with weight percentages,
the values of fatty acid phenacyl derivatives quantified by HPLC are
closer to mole percentages (Wood& Lee, 1983).
Methods
Culture conditions. The Flexibacter strains Inp2 and Inp3 used in this
study were isolated after continuous cultivation of the wild-type
Flexibacter strain Inp on high salinity medium (Intriago & Floodgate,
1991). Besides the differences in fatty acid composition, no nutritional,
physiological or morphological differences between the strains were
found. The culture conditions were as described by Intriago &
Floodgate (1991). The carbon source was 12% (w/v) sucrose, 0.5%
(w/v) glucose or 0.5% (w/v) soluble starch. Salinity was increased by
adding the artificial sea-water salts formulated by Reichenbach &
Dworkin (1981). The doubling time of all strains in each of the media
was 12 h. When the cultures reached exponential phase (about 40 h),
sodium [ 1-l4C]acetate [Amersham; specific radioactivity 50-60 mCi
mol-l (1.85-2-22 GBq mol-I)] was added to the medium to give a final
concentration of 1-0pCi ml-l (37 kBq ml-l) and incubated for 4 h
before harvesting the bacteria.
When either cAMP (1 mM, Aldrich) or cerulenin (5 pg ml-', Sigma)
were included in the incubation mixtures for Inp2, they were added at
the same time as the radioisotope.
When cAMP (1 mM) or the uncoupler 2,4-dinitrophenol (DNP;
I p ~ BDH)
;
were added to the culture medium of Inp3 they were
sterilized by filtration and added to the growth medium at the
beginning of the experiment.
Lipid extraction. Bacteria were harvested as described in Intriago &
Floodgate (1991). Lipids were extracted by the method of Bligh & Dyer
(1959), saponified with 2 ml5% (w/v) KOH in 50% (v/v) methanol in
water for 1 h at 100 "C, cooled and acidified with 6 M-HCl. The fatty
acids were extracted twice with 1 ml chloroform and dried under
nitrogen.
Gas-liquid chromatographic analysis. Fatty acids of Inp3 were
methylated and analysed as described in Intriago & Floodgate (1991).
Radio-HPLC analysis. Fatty acids of Inp2 were derivatized using a
technique adapted from that of Lam & Gruska (1985). Derivatization
was started by adding 20 p1 5 % (w/v) KOH in 50% (v/v) methanol in
water to the dried lipid sample, and then evaporating the methanol and
water using a stream of nitrogen gas. The fatty acid potassium salts so
formed were dissolved in 1 ml acetonitrile, plus 150 p1 pbromophenacyl bromide (Sigma) (50 mg ml-l) and 150 pl 18 crown 6 (dicyclohexano-18-crown-6) (Aldrich) (5 mg ml-I) dissolved in acetonitrile. The
reaction mixture was heated for 25 min at 85 "C. Excess reagent after
derivatization was eliminated by adding 2 ml water and 2 ml hexane to
the reaction vial and mixing vigorously. The hexane layer was removed
and placed in a second vial and evaporated using nitrogen gas. The
fatty acid methyl esters were redissolved in 100 pl acetonitrile, and
10 pl was injected into a Varian 2050 HPLC, fitted with a C18 ODs2 3p
150 cm x 4.6 mm column (Spherisorb, Alltech). An isocratic programme with acetonitrilefwater (85 :15, v/v) as mobile phase was used,
with a flow rate of 1-5mlmin-' equivalent to 120 bar, and was
monitored at a wavelength of 254 nm. The radioactive detector was a
Fatty acid biosynthesis in Flexibacter strain I p 2
The fatty acid composition of Flexibacter strain Inp2 was
broadly similar whether it was grown in 12% sucrose or
0.5 % glucose (cf. Tables 1 and 2). Both C 18 :2 and C 18 :3
Table 1. Percentage fatty acid composition and distribution
of radioactivity among fatty acids in Flexibacter strain Inp2
afer growth on 12% sucrose
Values represent two separate experiments, after correcting for
quenching and background.
Fatty
acid
14:O
14: 1
i15 : O
a15:O
15 :O
16 :0/18 :1
16: 1
18:2
18:3
Unknown
Composition
by mass (%I
3.5-1.7
4.8-1.2
4.6-5-1
53-50
0.0-0-0
8.1-7-8
61-5-57.2
8.7-7-3
34-34
0.6-0-4
Radioactivity (%)
3-9-59
44-45
0-0-0.0
0*0-0.0
0.0.0
25.2-32.2
53.5-54.2
8G-3.1
3.2-0.0
0.0-0.0
Total c.p.m. 12280-33210
Table 2. Percentage fatty acid composition and distribution
of radioactivity among fatty acids in Flexibacter strain Inp2
afer growth on 0.5% glucose
Values represent two separate experiments, after correcting for
quenching and background.
Fatty
acid
14:O
14: 1
i15 : O
a15 : O
15:O
16:0/18 : 1
16: 1
18:2
18:3
Unknown
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Composition
by mass (%)
3.1-2.8
7.0-1 1.1
44-7.2
8.140
0.0.0
8.1-1 3.0
56.8-55-5
7.3-6-9
3.440
1* 1-3.2
Radioactivity (%)
4.9-2.3
0-0-0.0
0-0-0.0
o*o-o.o
0.0-0.0
34.5-56-4
5 4 - 8 41*2
0*0*0
0.0-0.0
0.0.0
Total c.p.m. 9940-14060
Unsaturated fatty acid synthesis in Flexibactor
11 1
Table 3. Eflect of CAMP on fatty acid composition and
distribution of radioactivity among fatty acids in Flexibacter
strain Inp2 after growth on 12% sucrose
Table 5 . Efect of cerulenin on fatty acid composition and
distribution of radioactivity among fatty acids in Flexibacter
strain Inp2 after growth on 0.5% glucose
Values represent two separate experiments, after correcting for
quenching and background. cAMP (1 mM) was added together
with the radioactive precursor 4 h before harvesting the bacteria.
Values represent two separate experiments, after correcting for
quenching and background. Cerulenin ( 5 pg ml-l) was added
together with the radioactive precursor 4 h before harvesting the
bacteria.
Fatty
acid
Composition
by mass (%I
~~
14:O
14: 1
i15 :O
a15:O
15:O
16:0/18: 1
16: 1
18:2
18:3
Unknown
2-4-3-6
4.3-6-4
2-3-46
4-5-6.4
0.0.0
35.7-9.4
364-57-5
124-7.2
0.0-2.8
1-0-2-4
Radioactivity (%)
~~
~
36-5-20.9
0.0.0
0.0.0
0.0.0
0.0-0-0
48.1-36.0
14.2-37.3
0.0*0
0.0-0.0
0*0*0
Total c.p.m. 12400-15300
Table 4. Efect of cerulenin on fatty acid composition and
distribution of radioactivity among fatty acids in Flexibacter
strain Inp2 after growth on I2% sucrose
Values represent two separate experiments, after correcting for
quenching and background. Cerulenin ( 5 pg ml-l) was added
together with the radioactive precursor 4 h before harvesting the
bacteria.
Fatty
acid
14:O
14: 1
i15 :O
al5:O
15 :O
16:0/18 : 1
16: 1
18:2
18:3
Unknown
Composition
by mass (%)
2.7-3.3
10.9-7.3
5.147
94-7.0
0.0-0.0
8.1-1 1.9
544-54.6
4-0-4.6
2.9-3.1
2.7- 1*8
Radioactivity (%)
Fatty
acid
Composition
by mass <%)
14:O
14: 1
il5:O
a15:O
15:O
16:0/18: 1
16: 1
18:2
18:3
Unknown
3.5-2.1
8.8-3-7
5.8-2.1
0.0-4.0
0-0-3
3 1-7-32.6
3 4 - 4 41*5
13.1-10.6
04-1.3
2 - 61-7
Radioactivity (%)
26.5-1 1.6
1-7-1.0
8.9-2.8
0.0.0
0-0-1*8
43.9-49-0
16.0-2 1a8
24-7.9
0.0-2-6
o*o-o-o
Total c.p.m. 8450-6140
Addition of cerulenin to the sucrose medium resulted
in a marked decrease of fatty acid synthesis; radiolabelled acetate was incorporated only into the palmitic/
oleic acids fraction (Table 4). Cerulenin had a different
effect on cultures grown in glucose medium (cf. Tables 4
and 5). In contrast to bacteria grown in 12% sucrose,
incorporation of radioactive acetate into C16 :1 and total
fatty acids was only partly inhibited, and the precursor
was readily incorporated into both PUFAs (Table 5).
0.0.0
o*o.o
0.0*0
0.0-0.0
0.0-0.0
100*0-100~0
0.0-0.0
0.0-0
0.0-0.0
O~O-o*O
Total c.p.m. 1960-640
were present in bacteria grown in either medium.
However, none of the radiolabelled acetate appeared in
these acids in glucose-grown cells after incubation with
the precursor for 4 h, whereas a significant amount of
radioactivity was incorporated into these acids in
sucrose-grown cultures (Tables 1 and 2).
Addition of cAMP to the sucrose medium did not
affect bacterial growth, but prevented the biosynthesis of
PUFAs. The relative percentages of radiolabelled
material incorporated from acetate into myristic and
palmitic/oleic acids were increased compared with the
control whilst the incorporation into palmitoleic/oleic
acids was decreased (cf. Tables 1 and 3).
Eflect of osmotic strength on Flexibacter strain Inp3
A comparison of the fatty acid composition of Flexibacter strain Inp3 cultured in glucose medium (Table 6)
with that of strain Inp, described in Intriago & Floodgate
(1991), under the same conditions highlights the dissimilarities between these two strains : whereas PUFAs were
practically absent in Inp3, they represented up to 20% of
the total fatty acids in Inp. However, the data in Table 6
illustrates that synthesis of PUFAs was restored once
Inp3 was grown in a high osmotic strength medium such
as 12% sucrose or starch-Inp medium at 60p.p.t.
salinity.
Table 7 shows the fatty acid composition of Inp3
cultured in five different experiments, with starch-Inp
containing betaine as basic medium, and four individually added salts, at the concentrations given for the
artificial sea-water mixture described by Reichenbach &
Dworkin (198 1). In another experiment 30 g KC1 1-l
(approximately 0.4 M, final concentration) was added to
the medium to compare the effects of equal weight
concentrations of NaCl and KCl. Addition of NaCl
alone led to PUFA levels similar to those obtained with
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112
P . intriago
Table 6. Fatty acid composition of Flexibacter strain Inp3 afer growth on diflerent media
Values in parentheses represent means A 1 SD from two separate experiments. ND, Not detected.
Fatty acid composition (percentage of total fatty acids)
~~~
Starch
( 5 g 1-l)
Sucrose
(12%)
4.3(0.6)
0-2(0*1)
0*4(0*2)
25*0(1-2)
2*1(0-2)
7*9(1-0)
0-8(0-2)
0-4(0.6)
23.1 (0.8)
1*O(O-3)
60-8(0-2)
1*3(0.9)
0*8(0-3)
0*2(0-1)
0.2(0-0)
2*5(0*0)
57*9(0-1)
0.6(0*3)
0*9(0.0)
O.l(O*1)
0.2(0.1)
1*3(0-6)
1.6(0*3)
0.2(0.1)
0.4(0.2)
28.4(0-0)
0*5(0*2)
1.2(0.2)
13*3(1-5)
4.1(1-0)
10.0(1* 1)
2*2(0*3)
9*8(0*8)
l.O(O.2)
2.1 (0.8)
10.2(2*5)
Glucose
(5 g 1-l)
Fatty
acid
14 :O
i15: 1
a15:O
15:O
16:O
16: 109
16: 1 0 7
16: 105
18:O
18 : 109
18: 107
18 :206
18:303
20: 109
Unknown
ND
ND
ND
ND
ND
ND
ND
ND
ND
Sucrose (12%)
1 mM-CAMP
+
1*8(0*3)
4.4(0.1)
0.4(0-3)
0*8(0-5)
23*3(4*3)
0.2(0.3)
1.6(0-6)
43-5(2.4)
2-4(0*4)
5*2(2*1)
1.2(0*7)
2.3(0-9)
0-6(0-4)
ND
ND
Table 1. Fatty acid composition of Flexibacter strain Inp3
afer growth on starch-Inp medium plus salts
Flexibacter strain Inp3 was grown on starch-Inp medium
containing 10 mwbetaine together with individually added salts
at concentrations corresponding to those in the artificial sea-water
mixture described in Methods. m,Not detected.
Starch
(60P.P.t.1
2.2(0.6)
2.0(0*3)
0.5(0*2)
0.8(0.3)
28*5(0*4)
2*4(0*4)
2*1(0.1)
22.1 (4.7)
4-7(0.3)
6*8(0*7)
5.8( 1.1)
6*1(0*9)
2,0(0.2)
ND
ND
Starch (60p.p.t.)
1 mM-CAMP
Starch (60p.p.t.)
1 ~M-DNP
1*9(0*2)
6*3(0*
1)
0*3(0*4)
0.5 (0.7)
2 1*3(4*6)
2.3(0.4)
5*3(1.1)
0.9(0*3)
0*7(0*
1)
17*8(1* 1)
3*0(1-3)
45.6( 1 1*4)
4*9(1.1)
6*8(4*7)
2*2(3.1)
2*9(0*9)
0*7(0*0)
2-8(0*6)
59*3(0-5)
3.6(3.4)
1.5(0*1)
0.3(0.1)
0.9(0.5)
0.3(0.2)
+
ND
ND
ND
+
ND
ND
ND
(Table 6). Addition of cAMP reduced the proportion of
all acids with an 18-carbon chain length in Inp3. The
effects of the uncoupler DNP (1 p ~ and
) cAMP on the
fatty acid composition of Inp3 were similar (Table 6).
Although DNP did not prevent the growth of Inp3 it
reduced the growth rate approximately twofold.
~
Fatty acid composition (percentage of total fatty acids)
-~
~
Fatty
acid
NaCl
(30 g 1-l)
KCl
(0.7 g 1 - I )
~~~
MSSO4l
MgCl,
(1 1.2 g 1-l)
CaC1,
(1.0 g 1-I)
KCl
(30 g I-')
-
14 :O
i15:O
a15:O
15 :O
16:O
16: 109
16: 107
16: 105
18:O
18 :109
18: 107
18 :206
18 :303
1.2
0-9
0.6
6.5
0.6
39.0
21.7
21.3
19.9
2.3
62.6
1.3
1.4
59.4
2.8
1-9
1.5
55.7
2-4
2.7
0.3
1-4
0.6
ND
ND
ND
ND
18.0
25.2
5.8
ND
6.1
1.8
ND
ND
0.5
ND
0.4
ND
1.1
5.0
ND
ND
ND
ND
2.2
ND
1-7
6.9
ND
ND
ND
1.5
4.1
0.1
0.2
14.9
0.7
0.4
61.4
1.5
5.2
0.8
1.0
0.4
the full artificial sea-water complex. Both
MgSO,/MgCl, and CaCl, slightly increased the PUFA
content, whears no change was found when KCI was
added at either concentration.
Eflect of CAMP and DNP on Flexibacter strain inp3
Addition of cAMP to the culture medium lowered the
proportion of PUFAs in Flexibacter strain Inp3 growing
at a high salt concentration in 12% sucrose medium
Discussion
Intriago & Floodgate (1991) regarded the presence and
inverse relationship in Flexibacter strain Inp between the
two major fatty acids C 16 :1w5 and C 18 : 1w9 as evidence
for the presence of both the aerobic and anaerobic
pathways for synthesis of unsaturated fatty acids in this
bacterium. If the anaerobic pathway were present the
antibiotic cerulenin should inhibit synthesis by this
pathway. Fatty acid metabolism in Flexibacter strain
Inp2 was affected by cerulenin in two ways: firstly,
cerulenin inhibited fatty acid synthetase, and with it the
synthesis of C16:l; secondly, it enhanced PUFA
synthesis. These two effects could be the result of the
presence of both the aerobic and anaerobic pathways for
UFA synthesis. It also shows that as the flow of substrate
from one pathway rises, the flow of substrate from the
other is lowered. Wada et al. (1989) demonstrated the
presence of both pathways for fatty acid synthesis in
Pseudomonas E-3. The selectivity of cerulenin to inhibit
UFA synthesis by the anaerobic pathway, is well
documented (Buttke & Ingram, 1978; Wada et al., 1989).
The partial inhibition of C I6 :1 synthesis by cerulenin in
glucose medium may be due to either a more resistant
/?-keto-acyl synthase I present in bacteria grown in
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Unsaturated fatty acid synthesis in Flexibactor
glucose, or the palmitoleate detected was an isomer other
than C16:lw5, and was synthesized by the aerobic
pathway.
The results of Intriago & Floodgate (1991), together
with these from the present study, show that high
osmotic strength stimulates PUFA production in Flexibacter Inp and related strains, and that this is prevented
by addition of cAMP to the medium. Piovanti &
Lazdunski (1975) demonstrated that media with high
osmotic strength, such as 12% sucrose, caused a fourfold
decrease in the intracellular cAMP level in E. coli, and
Brownlie et al. (1985) found that a high NaCl concentration in the medium inhibited adenylate cyclase in
Bordetella pertussis. Thus it seems possible that high
osmotic strength media lower the intracellular levels of
cAMP in Flexibacter Inp strains, resulting in the
observed increase in PUFA content. However, it is not
clear whether the effect of cAMP on Inp2 and Inp3 was
involved in facilitating the transcription of genes
encoding for proteins, as has been reported for other
organisms (Buettner et al., 1973; Botsford, 1981 ; Roy et
al., 1988), or in the activation of a CAMP-dependent
protein kinase. Although protein kinases have been
thought to be absent in prokaryotes (Dadssi & Cozzone,
1985), some exceptions have been found (Muller et al.,
1985; Saha et al., 1988). More recently, Kinney et al.
(1990) demonstrated regulation of phospholipid biosynthesis by a cAMP kinase in S . cereuisiae.
The present study has demonstrated that PUFA
synthesis is restored in the normally PUFA-deficient
Flexibacter strain Inp3 by increasing the osmotic
strength in the medium either with ionic solutes such as
NaCl or non-ionic ones such as sucrose. Nevertheless, it
is clear from the data that NaCl and sucrose are more
effective in increasing the proportion of PUFAs than
was KCl. However, whilst sea-water medium containing
sucrose gives an extra 0-225g-mol osmosity units to that
of the sea water (0.473 g-mol), 30 g 1-1 of both NaCl and
KCl gives 0.523 g-mol and 0.404 g-mol osmosity units
respectively (Weast, 1987). Hence it can be concluded
that osmotic strength is not the only factor controlling
PUFA biosynthesis.
Addition of the uncoupler DNP has been shown to
raise the cAMP level in the yeast Saccharomyces
cerevisiae (Thevelein et al., 1987). Fatty acid analysis of
protonophore-resistant bacteria has revealed variation
between species. Thus, whilst unsaturated fatty acids
decreased in Bacillus subtilis (Krulwich et al., 1987), they
increased in E. coli (Herring et al., 1985). Furthermore,
Kogure & Tokuda (1986) showed that the growth of
marine bacteria can depend on either the proton- or
sodium-motive force. On this basis, it may be suggested
that the inhibition by DNP of PUFA synthesis in
Flexibacter Inp3, as seen in the present study, was caused
113
by an increase in cellular CAMP, which in turn was
regulated by changes in membrane electrochemical
potenti a1.
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